Technical Field
The present invention relates generally to cell culture conditions, media, and culture platforms for culturing stem cells, including feeder-free conditions for generating and culturing human induced pluripotent stem cells (iPSCs).
Description of Related Art
The application of pluripotent stem cell biology opens new doors for regenerative medicine. The derivation of human embryonic stem cells (hESC) by culturing pre-implantation blastocysts in cocktails of growth factors has led to many promising cell therapy approaches where the expanded self renewing population of cells can be differentiated to the therapy-relevant cell lineage in vitro or in vivo. In a further application of ESC biology and by using pre-implantation genetic analysis it has been possible to derive ESC lines from several genetic disease backgrounds, and thus, model these diseases in the tissue culture dish. There are, however, some limitations to ESC technology: the range of genetic backgrounds from which ESC can be derived are both technically and politically limited, the genetic background of the ESCs are not always known and the use of ESC-derived cell therapy is essentially an allograft, running the same rejection risks as traditional tissue/organ transplants.
In a major advance, pluripotent cell populations were generated from adult, terminally differentiated cells: such derived cells are called induced pluripotent stem cells (iPSC). iPSC technology allows cells from any donor to be reprogrammed into a pluripotent, self renewing state and thus allow the expansion of a homogeneous population of cells from any genetic background. iPSCs overcome ethical considerations pertaining to ESCs and can be used to derive models of any genetic human disease for high throughput drug screening or hepatocytes and cardiomyocytes for xenobiotic drug toxicity screening. Further, iPSCs may ultimately result in cell therapies generated from the patient's own cells in an autologous transplantation that may prevent graft rejection. Expression and differentiation analysis has shown iPSCs to be very close to ESCs at the molecular level with variations between clonal iPSC cultures of similar magnitude to those seen when comparing multiple ESC lines.
iPSCs have generally been generated by ectopic expression of several key genes shown to be required for full reprogramming, namely combinations of: Oct4, Sox2, Klf4, c-Myc, Lin28 and Nanog. iPSCs were originally generated using integrating viral systems to express key transcription factors. Retroviral and lentiviral systems including polycistronic and inducible systems have now been successfully employed in iPSC generation. However, permanent genomic changes due to insertional mutagenesis and the potential for exogenous gene reactivation post iPSC differentiation may present potential problems for subsequent drug screening and therapeutic applications of cells generated by these methods. Indeed, significant differences between iPSC clones generated using the same viral systems have been reported, with a large percentage of clones forming tumors in rodents when transplanted as differentiated neurospheres. Research suggests that iPSCs generated using the same viral methods may behave differently once differentiated. Differences in ectopic gene integration site may result in different insertional mutagenesis and epigenetic regulation of transgene expression. For iPSC generation methods that include integrating systems, many clones may need to be derived and screened to identify those that are stable in both pluripotent and differentiated states. Thus, a method for the rapid derivation of clonal iPSCs from a given donor cell source would be beneficial. The use of non-integrating systems for iPSC generation such as adenoviral or episomal transient expression have also been demonstrated, albeit with lower efficiency. These systems may overcome safety and stability issues in iPSC generation, however there is a potential for genomic integration when using any DNA-based reprogramming method and this would need to be assessed prior to their use in development of an iPSC-derived cell therapy.
Excisable viral systems and genome wide expression profiling show that iPSCs with integrated expression cassettes are less like ESCs than the same clones with the viral factors excised. Further, protein-only reprogramming has now been demonstrated in which the most commonly used transcription factors were expressed in E. coli as fusion proteins with cell penetrating peptides. Multiple doses of the purified proteins were applied to murine fibroblasts resulting in iPSC generation. The efficiency of reprogramming using this protein-only system was very low. This may be due to the efficiency of the protein transduction, the specific activity of the protein and/or the stability of the proteins.
The process of differentiated cell reprogramming by the ectopic expression of pluripotency genes or their introduction via protein transduction or mRNA requires several months and the knowledge of a skilled stem-cell biologist. The identification of reprogrammed cells is initially by eye: screening for of ESC-like colony morphology. Such colonies must be picked by hand, are usually mechanically passaged and expanded. The introduction of the pluripotency factors also produces transformed cell colonies as well as incompletely reprogrammed cells. A researcher may be able to identify the true iPSC colonies from the background of transformed cells, but this is not an efficient process. Further characterization and recognition as a true pluripotent population is then required and usually includes immunocytochemistry staining for markers of pluripotency, gene expression and epigenetic analysis and the ability of the pluripotent population to differentiate to the three germ layers (ectoderm, mesoderm and endoderm). Once pluripotent cells are identified and selected, such cells are generally grown as colonies and require manual passaging by picking and mechanically dissociating cells prior to replating to maintain cells long-term.
Embryonic stem cells derived from various pre- and post-implantation stages display distinct states of pluripotency. For example, cells derived from the inner cell mass of a blastocyst are considered more “naïve” and have key properties that are quite different from the postimplantation derived cells that are considered more “primed” with higher propensity to randomly differentiate. Naïve cells appear to be in a more “grounded state” and do not require extrinsic signaling to maintain their undifferentiated status. On the other hand, primed cells require extrinsic signaling of key cytokines including TGFβ, Activin and bFGF and are quite dependent on the ERK/MAPK cellular pathway for maintaining their undifferentiated status.
Improvements to the iPSC generation process could dramatically lower the technical barriers, speed-up the process and enable subsequent scale-up and differentiation of cells for industrial applications of the technology such as drug screening and cell therapy. Methods for more efficient production of iPSCs without the use of exogenous material, and more efficient identification and selection of reprogrammed cells are required. Methods of generating iPSCs that promote the naïve state of human pluripotent stem cells would be greatly advantageous for future applications in regenerative medicine, such as disease correction, directed differentiation and manufacturing-scale expansion. Further, methods for more efficient production of iPSCs in defined culture conditions that enable single cell passage and scalability are required.